Production of M2000 (ß-d-mannuronic acid) and its therapeutic effect on experimental nephritis.

UNLABELLED: The present research introduces the method of Production of M2000 (ß-d-mannuronic acid) and its therapeutic effect on experimental model of nephritis. M2000 was produced using enzymatic and chemical procedure on prepared alginate from Pseudomonas fluorescens. The experimental glomerulonephritis was induced in rats by a subcutaneous immunization and daily intravenous administration of bovine serum albumin (BSA). M2000 solution (30mg/kg) was administered intraperitoneally at regular 48-h intervals for 4 weeks. Onset of treatment was day 56. Urinary protein was measured weekly and serum anti-BSA antibody was assessed by ELISA method at different intervals. Animals were killed on day 84 and blood samples and kidney specimens were obtained. Serum (creatinine, BUN, cholesterol, and triglyceride) and urine (protein, urea, and creatinine) determinants were measured at the time of sacrifice. Kidney specimens were processed for light and immunofluorescent microscopic examination. The fibrosarcoma cell line was used for assaying tolerability and matrix metalloproteinase type 2 (MMP-2) activity. MMP-2 activity was assessed using zymography. Our data showed that M2000 therapy could significantly reduce the urinary protein excretion in treated rats versus non-treated controls. Anti-BSA antibody titer was lower in treated rats than in controls at the 12th experimental week. PMN infiltration and glomerular immune complex deposition was less intense in treated rats than in controls. Cytotoxicity analysis of M2000 showed a much higher tolerability compared with other tested drugs (diclofenac, piroxicam and dexamethasone). The inhibitory effect of M2000 in MMP-2 activity was significantly greater than that of dexsamethasone and of piroxicam at a concentration of 200µg/ml. Moreover, the toxicological study revealed that M2000 had no influence on serum (BUN, creatinine, triglyceride and cholesterol) determinants, urinary protein excretion and glomerular histology in healthy group receiving drug. CONCLUSIONS: In this research, for the first time we introduced the procedure of production of M2000 (ß-d-mannuronic acid) and our data suggest that treatment with M2000, as a novel anti-inflammatory drug can reduce proteinuria, diminish antibody production and suppress the progression of disease in experimental model of glomerulonephritis.

Biochemical analysis of alginate biosynthesis protein AlgX from Pseudomonas aeruginosa: purification of an AlgX-MucD (AlgY) protein complex.

AlgX was found to be an essential protein for alginate biosynthesis, but its function is unknown. In this study, an isogenic, marker-free algX-knock out mutant was generated. In-frame fusions of algX with phoA and lacZ were analysed, respectively. No LacZ-activity was detected, but the PhoA fusion showed alkaline phosphatase activity. These data indicated that the C-terminus of AlgX is located in the periplasm, but is not required for protein function. Accordingly, AlgX with C-terminal fusion of strep tag II restored alginate production in the algX-negative mutant and was purified under native conditions from periplasmic and crude cell extracts, respectively. AlgX was identified by MALDI/TOF-MS analysis of tryptic peptides. TritonX-100 mediated solubilisation of cytoplasmic membrane and subsequent strep tag II affinity chromatography led to purification of an AlgX-MucD (AlgY) protein complex as identified by MALDI/TOF-MS analysis. This data suggested a protein-protein interaction between AlgX and MucD (AlgY) with a 1:1 stoichiometry. Thus AlgX might exert its function via interaction with MucD (AlgY). Immunoelectron microscopic localisation of AlgX-strep tag II suggested a localisation close to the cytoplasmic membrane.

Resolution of sirolimus-induced pneumonitis after conversion to everolimus.

BACKGROUND: Sirolimus-induced pneumonitis usually requires the complete cessation of sirolimus. Herein we have reported five cases of recovery from sirolimus pneumonitis after conversion from sirolimus to everolimus. PATIENTS: All five cases were comparable with regard to their clinical conditions. The ages were between 46 and 64 years. They had received kidney transplants 3 to 18 years earlier. In four cases, the reason for sirolimus therapy was toxicity due to calcineurin inhibitors on a transplant biopsy; three of the patients also displayed malignant tumors: renal cell carcinoma, spinocellular carcinoma, or melanoma. Their serum creatinine levels were elevated between 150 and 350 micromol/L. In all five cases, bronchoscopy disclosed lymphocytic pneumonitis and bronchiolitis obliterans. The immunosuppressive co-medications were prednisolone in three, azathioprine in one, and mycophenolate mofetil in four cases. The previous sirolimus dose was 1 to 4 mg/day, with sirolimus trough levels between 5 and 12 ng/mL. The patients were switched to everolimus at doses between 1 x 0.25 and 2 x 0.75 mg/day to achieve trough concentrations between 3 and 8 ng/mL. Pulmonary symptoms and radiological findings resolved completely within 1 to 4 weeks. CONCLUSION: Everolimus is more hydrophilic by virtue of differing from sirolimus by one hydroxyl group. Sirolimus-induced pneumonitis improved after conversion to everolimus.

Protective T Cell and Antibody Immune Responses against Hepatitis C Virus Achieved Using a Biopolyester-Bead-Based Vaccine Delivery System.

Hepatitis C virus (HCV) infection is a major worldwide problem. Chronic hepatitis C is recognized as one of the major causes of cirrhosis, hepatocellular carcinoma, and liver failure. Although new, directly acting antiviral therapies are suggested to overcome the low efficacy and adverse effects observed for the current standard of treatment, an effective vaccine would be the only way to certainly eradicate HCV infection. Recently, polyhydroxybutyrate beads produced by engineered Escherichia coli showed efficacy as a vaccine delivery system. Here, an endotoxin-free E. coli strain (ClearColi) was engineered to produce polyhydroxybutyrate beads displaying the core antigen on their surface (Beads-Core) and their immunogenicity was evaluated in BALB/c mice. Immunization with Beads-Core induced gamma interferon (IFN-γ) secretion and a functional T cell immune response against the HCV Core protein. With the aim to target broad T and B cell determinants described for HCV, Beads-Core mixed with HCV E1, E2, and NS3 recombinant proteins was also evaluated in BALB/c mice. Remarkably, only three immunization with Beads-Core+CoE1E2NS3/Alum (a mixture of 0.1 µg Co.120, 16.7 µg E1.340, 16.7 µg E2.680, and 10 µg NS3 adjuvanted in aluminum hydroxide [Alum]) induced a potent antibody response against E1 and E2 and a broad IFN-γ secretion and T cell response against Core and all coadministered antigens. This immunological response mediated protective immunity to viremia as assessed in a viral surrogate challenge model. Overall, it was shown that engineered biopolyester beads displaying foreign antigens are immunogenic and might present a particulate delivery system suitable for vaccination against HCV.

Alginate lyase from Pseudomonas aeruginosa CF1/M1 prefers the hexameric oligomannuronate as substrate.

In order to investigate the catalytic properties of alginate lyase from Pseudomonas aeruginosa CF1/M1, a clinical isolate, regarding the capability to perform beta-elimination on oligomannuronates of defined length (2-9), the alginate lyase was purified from periplasmic extracts. A purification method for unsaturated and saturated oligomannuronates applying anionic exchange chromatography on a FPLC apparatus was established. The alginate lyase showed the highest activity, when hexamers were provided as substrate. This indicated that the alginate lyase best accommodates a chain of six alginate residues in the active center. As a minimum chain length, the pentameric oligomannuronate was still accepted as substrate. Mannuronate oligomers shorter than the pentamer were not accepted as substrate for alginate lyase. Furthermore, oligomer pattern analysis of polymannuronate which was subjected to beta-elimination by alginate lyase revealed that the trimer is the most abundant oligomer. These data indicated that beta-elimination and cleavage occurred at mannuronic acid residue no. 3 of the accommodated hexameric alginate chain.

Functional expression of the PHA synthase gene phaC1 from Pseudomonas aeruginosa in Escherichia coli results in poly(3-hydroxyalkanoate) synthesis.

The potential of the production of polyhydroxyalkanoates (PHA), consisting of medium-chain-length (MCL) hydroxyfatty acids (C5-C14), in recombinant Escherichia coli was investigated. E. coli mutants affected in fatty acid degradation and fatty acid de novo synthesis were employed. We established the functional expression of the Pseudomonas aeruginosa PHA synthase gene phaC1. The coding region of phaC1 was subcloned via PCR into vector pBluescript SK-. The resulting plasmid pBHR71 enabled functional expression of phaC1 under lac promoter control and conferred synthesis and accumulation of PHA to various strains of E. coli. PHA synthesis was analysed with respect to the carbon source in various E. coli fad and fab mutants. This study provided evidence that intermediates of the fatty acid beta-oxidation can be directed to PHA synthesis and that 3-hydroxydecanoyl-CoA is the main substrate for PHA synthase PhaC1 from P. aeruginosa. The E. coli fadB mutant LS1298 containing plasmid pBHR71 and cultivated in LB medium containing 0.5% (w/v) decanoate revealed the strongest accumulation of PHA contributing to about 21% of the cellular dry weight, which was composed of 2.5 mol% 3-hydroxyhexanoate, 20 mol% 3-hydroxyoctanoate, 72.5 mol% 3-hydroxydecanoate and 5 mol% 3-hydroxydodecanoate.

Analysis of in vivo substrate specificity of the PHA synthase from Ralstonia eutropha: formation of novel copolyesters in recombinant Escherichia coli.

In order to investigate the in vivo substrate specificity of the type I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha, we functionally expressed the PHA synthase gene in various Escherichia coli mutants affected in fatty acid beta-oxidation and the wild-type. The PHA synthase gene was expressed either solely (pBHR70) or in addition to the R. eutropha genes encoding beta-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase comprising the entire PHB operon (pBHR68) as well as in combination with the phaC1 gene (pBHR77) from Pseudomonas aeruginosa encoding type II PHA synthase. The fatty acid beta-oxidation route was employed to provide various 3-hydroxyacyl-CoA thioesters, depending on the carbon source, as in vivo substrate for the PHA synthase. In vivo PHA synthase activity was indicated by PHA accumulation and substrate specificity was revealed by analysis of the comonomer composition of the respective polyester. Only in recombinant E. coli fad mutants harboring plasmid pBHR68, the R. eutropha PHA synthase led to accumulation of poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (poly(3HB-co-3HO)) and poly(3HB-co-3HO-co-3-hydroxydodecanoate (3HDD)), when octanoate and decanoate or dodecanoate were provided as carbon source, respectively. Coexpression of phaC1 from P. aeruginosa indicated and confirmed the provision of PHA precursor via the beta-oxidation pathway and led to the accumulation of a blend of two different PHAs in the respective E. coli strain. These data strongly suggested that R. eutropha PHA synthase accepts, besides the main substrate 3-hydroxybutyryl-CoA, also the CoA thioesters of 3HO and 3HDD.

The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources.

We recently identified the phaG(Pp) gene encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase in Pseudomonas putida, which directly links the fatty acid de novo biosynthesis and polyhydroxyalkanoate (PHA) biosynthesis. An open reading frame (ORF) of which the deduced amino acid sequence shared about 57% identity with PhaG from P. putida was identified in the P. aeruginosa genome sequence. Its coding region (herein called phaG(Pa)) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The resulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phaG(Pp)-negative mutant P. putida PhaG(N)-21. The PHA was composed of 5 mol% 3-hydroxydodecanoate, 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate and 5 mol% 3-hydroxyhexanoate. Furthermore, an isogenic phaG(Pa) knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaG(Pa) mutant did not show any difference in growth rate, but PHA accumulation from gluconate was decreased to about 40% of wild-type level, whereas from fatty acids wild-type level PHA accumulation was obtained. These data suggested that PhaG from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity and strongly enhances the metabolic flux from fatty acid de novo synthesis towards PHA(MCL) synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known.

Synthesis of poly(3-hydroxyalkanoates) in Escherichia coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa: comparison of PhaC1 and PhaC2.

In order to obtain functional expression of PHA synthase gene phaC2 from Pseudomonas aeruginosa in Escherichia coli, the coding region of phaC2 was subcloned, including the ribosomal binding site, into pBluescript SK- collinear to the lac promoter. This plasmid pBHR71-C2 enabled functional expression of phaC2 in E. coli LS1298 (fadB) under lac promoter control, leading to PHA accumulation, when grown in LB medium containing 0.5% (w/v) of various fatty acids (C8-C14). The strongest accumulation of PHA was observed, when dodecanoate was provided as carbon source, and PHA contributed to 15% of cell dry weight, which was composed of 35 mol% 3-hydroxydodecanoate, 60 mol% 3-hydroxydecanoate and 5 mol% 3-hydroxyoctanoate. Plasmid pBHR78, which contained both genes phaC1 and phaC2 from P. aeruginosa under lac promoter control in pBluescript SK- led in E. coli LS1298 to PHA accumulation, which contributed to 13% of cell dry weight, when cells were grown on decanoate. Only slight differences in PHA composition compared with either PhaC1 or PhaC2 were obtained. The weight average molecular masses of PHA purified from decanoate-grown cells of E. coli LS1298 expressing PhaC1 or PhaC2 alone or both PHA synthases, were 106 x 10(3), 70 x 10(3) or 67 x 10(3), respectively. This study clearly demonstrated that both PHA synthases from P. aeruginosa exhibit very similar properties resulting in similar extent of PHA accumulation, similar composition and molecular mass, when expressed in E. coli and that fatty acid beta-oxidation provides substrates for both PHA synthases.

Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition of fatty acid beta-oxidation by acrylic acid.

Heterologous expression of the phaC1 gene from Pseudomonas aeruginosa, which encodes one of the polyhydroxyalkanoic acid synthases, in Escherichia coli impaired in fatty acid beta-oxidation results in polyhydroxyalkanoic acid accumulation when cells were cultivated on fatty acids. We evaluated the application of the fatty acid beta-oxidation inhibitor acrylic acid as a tool to channel intermediates of beta-oxidation to polyhydroxyalkanoic acid synthesis. Various E. coli strains affected in fatty acid metabolism and the wild-type strain harboring plasmid pBHR71 were analyzed with respect to polyhydroxyalkanoic acid accumulation in the presence of acrylic acid. The E. coli fadR mutant RS3097 revealed the strongest polyhydroxyalkanoic acid accumulation. The optimum inhibitory concentration of acrylic acid was 0.24 mg ml-1 and caused efficient channeling of intermediates of beta-oxidation to polyhydroxyalkanoic acid synthesis. Under these conditions and grown on decanoate E. coli RS3097 harboring plasmid pBHR71 revealed a polyhydroxyalkanoic acid accumulation contributing to about 60% of cellular dry weight.

Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis.

Polyhydroxyalkanoic acids (PHA) represent a complex class of storage polyesters that are synthesized by a wide range of different gram-positive and gram-negative bacteria as well as by some Archaea and that are deposited as insoluble cytoplasmic inclusions. PHA synthases, which are the key enzymes for PHA biosynthesis, have been characterized in much detail. At present 42 PHA synthase structural genes from 38 different bacteria have been cloned, and from 30 genes the nucleotide sequences were obtained. The strategies successfully employed to clone these genes and the current knowledge on the organization of the PHA synthase genes and other genes encoding proteins related to PHA metabolism will be compiled. In addition, the primary structures of the 30 PHA synthases were aligned and analyzed with respect to highly conserved amino acids and biochemical features. The direction, in which research should proceed, in order to increase our knowledge on biosynthesis of PHAs and to utilize this knowledge for the development of technically and economically feasible processes for the production of these polyesters will be outlined.

Halopiger aswanensis sp. nov., a polymer-producing and extremely halophilic archaeon isolated from hypersaline soil.

Strain 56(T) was isolated from a hypersaline soil in Aswan (Egypt). Cells were pleomorphic rods. The organism was neutrophilic, motile and required at least 1.7 M (10 % w/v) NaCl, but not MgCl(2), for growth; optimal growth occurred at > or =3.8 M (> or =22.5 %) NaCl. The strain was thermotolerant with an optimum temperature for growth of 40 degrees C, although growth was possible up to 55 degrees C. The G+C content of the DNA of the novel strain was 67.1 mol%. 16S rRNA gene sequence analysis revealed that strain 56(T) was a member of the phyletic group defined by the family Halobacteriaceae, showing the highest similarity to Halopiger xanaduensis SH-6(T) (99 %) and the next highest similarity of 94 % to other members of the family Halobacteriaceae. DNA-DNA hybridization revealed 27 % relatedness between strain 56(T) and Hpg. xanaduensis SH-6(T). Polar lipid analysis revealed the presence of the bis-sulfated glycolipid S(2)-DGD-1 as the sole glycolipid and the absence of the glycerol diether analogue phosphatidylglycerosulfate. Both C(20 x 20) and C(20 x 25) core lipids were present. Strain 56(T) accumulated large amounts of polyhydroxybutyrate and also secreted an exopolymer. Physiological and biochemical differences suggested that Hpg. xanaduanesis and strain 56(T) were sufficiently different to be separated into two distinct species. It is suggested that strain 56(T) represents a novel species of the genus Halopiger , for which the name Halopiger aswanensis sp. nov. is proposed. The type strain is strain 56(T) (=DSM 13151(T)=JCM 11628(T)).

Therapeutic approach by a novel designed anti-inflammatory drug, M2000, in experimental immune complex glomerulonephritis.

UNLABELLED: The therapeutic efficacy of novel designed nonsteroidal anti-inflammatory drug, M2000 (beta- D- mannuronic acid) on experimental immune complex glomerulonephritis was evaluated. Bovine serum albumin (BSA) nephritis was induced in rats by a subcutaneous immunization and daily intravenous administration of BSA. M2000 solution (30 mg/kg) was administered intraperitoneally at regular 48-hr intervals for 4 weeks. Onset of treatment was day 56. Urinary protein was measured weekly and serum anti-BSA antibody was assessed by ELISA method at different intervals. Animals were killed on day 84 and blood samples and kidney specimens were obtained. Serum (creatinine, blood urea nitrogen, cholesterol, and triglyceride) and urine (protein, urea, and creatinine) determinants were measured at the time of sacrifice. Kidney specimens were processed for light and immunofluorescent microscopic examination. The fibrosarcoma cell line was used for assaying tolerability and matrix metalloproteinase type 2 (MMP-2) activity. MMP-2 activity was assessed using zymography. Our data showed that M2000 therapy could significantly reduce the urinary protein excretion in treated rats versus non-treated controls. Anti-BSA antibody titer was lower in treated rats than in controls at the 12th experimental week. Polymorphonuclear neutrophil leukocytes infiltration and glomerular immune complex deposition were less intense in treated rats than in controls. Cytotoxicity analysis of M2000 showed a much higher tolerability compared with other tested drugs (diclofenac, piroxicam and dexamethasone). The inhibitory effect of M2000 in MMP-2 activity was significantly greater than that of dexsamethasone and of piroxicam at a concentration of 200 microg/ml. Moreover, the toxicological study revealed that M2000 had no influence on serum (BUN, creatinine, triglyceride and cholesterol) determinants, urinary protein excretion and glomerular histology in healthy group receiving drug. CONCLUSIONS: These findings suggest that treatment with M2000 can reduce proteinuria, diminish antibody production, and suppress the progression of disease in a rat model of immune complex glomerulonephritis.

Treatment of experimental arthritis with M2000, a novel designed non-steroidal anti-inflammatory drug.

The current study was planned to explore the therapeutic potency of M2000 (beta-D-mannuronic acid), a novel designed non-steroidal anti-inflammatory drug (NSAID) in adjuvant-induced arthritis model. Arthritis was induced in Lewis rats by a single intradermal injection (0.1 ml) of heat-killed Mycobacterium tuberculosis (0.3 mg) in Freund's incomplete adjuvant into the right footpad. Fourteen days after injection of adjuvant, the contralateral left footpad volume was measured. The animals with paw volumes 0.37 ml greater than normal paws were then randomized into treatment groups. Orally and intraperitoneally administrations of test drugs (M2000, 40/mg/kg/day and indomethacin, 2/mg/kg/day) were started on day 15 post-adjuvant injection and continued until final assessment on day 25. The left hind limb was removed for histological evaluation. The WEHI-164 cell line was used for assaying tolerability and matrix metalloproteinase type 2 (MMP-2) activity. MMP-2 activity was assessed using zymography. Pharmacotoxicology study was carried out on animal models based on the evaluation of serum and urine determinants, histology of kidney, gastrointestinal tolerability and body temperature. Results showed that the orally administration as well as intraperitoneally injection of M2000 to arthritic rats induced a significant reduction in paw oedema. Histopathological assessment showed a reduced inflammatory cells infiltrate in joints of treated rats, as well as the number of osteoclasts present in the subchondral bone, tissue oedema and bone erosion in the paws were markedly reduced following M2000 therapy. Cytotoxicity analysis of M2000 showed a much higher tolerability compared with other tested drugs (diclofenac, piroxicam and dexamethasone). The inhibitory effect of M2000 in MMP-2 activity was significantly greater than that of dexamethasone and of piroxicam at a concentration of 200 microg/ml. Moreover, the toxicological study revealed that M2000 had no influence on serum (blood urea nitrogen, creatinine, triglyceride and cholesterol) and urine (urea and urinary protein excretion) determinants, glomerular histology and body temperature in normothermic rats and had no ulcerogenic effects on rats' stomach. Our data show that M2000, as a novel NSAID, could be strongly suggested as the safest anti-inflammatory drug for long-term administration.

Sodium alginate as a novel therapeutic option in experimental colitis.

The potential therapeutic effect of low-viscosity sodium alginate (LVA) was studied in a rat model of acute colitis induced by intracolonic administration of acetic acid. This experimental model produced a significant ulcerative colitis. Induction of colitis also significantly enhanced the serum and colonic mucosal cytokine (IL-6 and TNF-alpha) and eicosanoid (LTB4 and PGE2) levels, which paralleled with the severity of colitis. LVA solution was administered orally as drinking water at concentration of 0.5% (W/V) for 1 week. The tolerability and inhibitory effect of LVA on matrix metalloproteinase-2 (MMP-2) were tested using WEHI-164 cell line and zymography method. The results showed that LVA therapy is able to significantly reduce colonic damage score, histological lesion, serum and colonic mucosal IL-6, TNF-alpha, LTB4 and PGE2 levels in treated group compared with nontreated controls. Moreover, in vitro examinations revealed that treatment with LVA could diminish MMP-2 activity. It is concluded that LVA is able to suppress acetic acid-induced colitis in rats. Some of the action of LVA may be associated with its inhibitory effects on cytokine and eicosanoid production and MMP-2 activity. Our data suggest that LVA could potentially be a novel therapeutic option for inflammatory bowel disease.

Bioinformatic tools for DNA/protein sequence analysis, functional assignment of genes and protein classification.

The development of efficient DNA sequencing methods has led to the achievement of the DNA sequence of entire genomes from (to date) 55 prokaryotes, 5 eukaryotic organisms and 10 eukaryotic chromosomes. Thus, an enormous amount of DNA sequence data is available and even more will be forthcoming in the near future. Analysis of this overwhelming amount of data requires bioinformatic tools in order to identify genes that encode functional proteins or RNA. This is an important task, considering that even in the well-studied Escherichia coli more than 30% of the identified open reading frames are hypothetical genes. Future challenges of genome sequence analysis will include the understanding of gene regulation and metabolic pathway reconstruction including DNA chip technology, which holds tremendous potential for biomedicine and the biotechnological production of valuable compounds. The overwhelming volume of information often confuses scientists. This review intends to provide a guide to choosing the most efficient way to analyze a new sequence or to collect information on a gene or protein of interest by applying current publicly available databases and Web services. Recently developed tools that allow functional assignment of genes, mainly based on sequence similarity of the deduced amino acid sequence, using the currently available and increasing biological databases will be discussed.

Bacterial alginates: biosynthesis and applications.

Alginate is a copolymer of beta-D-mannuronic acid and alpha-L-guluronic acid (GulA), linked together by 1-4 linkages. The polymer is a well-established industrial product obtained commercially by harvesting brown seaweeds. Some bacteria, mostly derived from the genus Pseudomonas and belonging to the RNA superfamily I, are also capable of producing copious amounts of this polymer as an exopolysaccharide. The molecular genetics, regulation and biochemistry of alginate biosynthesis have been particularly well characterized in the opportunistic human pathogen Pseudomonas aeruginosa, although the biochemistry of the polymerization process is still poorly understood. In the last 3 years major aspects of the molecular genetics of alginate biosynthesis in Azotobacter vinelandii have also been reported. In both organisms the immediate precursor of polymerization is GDP-mannuronic acid, and the sugar residues in this compound are polymerized into mannuronan. This uniform polymer is then further modified by acetylation at positions O-2 and/or O-3 and by epimerization of some of the residues, leading to a variable content of acetyl groups and GulA residues. In contrast, seaweed alginates are not acetylated. The nature of the epimerization steps are more complex in A. vinelandii than in P. aeruginosa, while other aspects of the biochemistry and genetics of alginate biosynthesis appear to be similar. The GulA residue content and distribution strongly affect the physicochemical properties of alginates, and the epimerization process is therefore of great interest from an applied point of view. This article presents a survey of our current knowledge of the molecular genetics and biochemistry of bacterial alginate biosynthesis, as well as of the biotechnological potential of such polymers.

Membrane topology of the outer membrane protein OprH from Pseudomonas aeruginosa: PCR-mediated site-directed insertion and deletion mutagenesis.

The 21-kDa outer membrane protein OprH from Pseudomonas aeruginosa is overexpressed under Mg2+ starvation conditions and when overproduced causes resistance to polymyxin B, gentamicin, and EDTA. By circular dichroism analysis, OprH revealed a calculated beta-sheet structure content of 47.3%. PCR-based site-directed deletion and epitope insertion mutagenesis was used to test a topological model of OprH as an eight-stranded beta-barrel. Three permissive and seven nonpermissive malarial epitope insertion mutants and four permissive and four nonpermissive deletion mutants confirmed the general accuracy of this model. Thus, OprH is the smallest outer membrane protein to date to be confirmed as a beta-stranded protein.

Polyhydroxybutyrate biosynthesis in Caulobacter crescentus: molecular characterization of the polyhydroxybutyrate synthase.

Caulobacter crescentus was investigated with respect to polyhydroxybutyrate (PHB) biosynthesis. Polyhydroxyalkanoate (PHA) accumulation contributing to approximately 18% of the cell dry weight was obtained in the presence of glucose. Gas chromatography-mass spectrometry and gel permeation chromatography of the purified PHA showed that this polyester was solely composed of 3-hydroxybutyrate and had a weight average molar mass of 5.5 x 10(5) g mol(-1) and a polydispersity of 1.6. An ORF encoding a conserved, hypothetical protein which shared approximately 47% identity with the PHB synthase from Azorhizobium caulinodans was identified within the complete C. crescentus genomic sequence. This putative C. crescentus PHB synthase gene, phaC, consisted of a 2019 nt stretch of DNA (encoding 673 aa residues), which encoded a PHB synthase with a molecular mass of approximately 73 kDa. This is currently the largest PHA synthase identified. The phaC coding region was subcloned into vector pBBR1-JO2 under lac promoter control. The resulting plasmid, pQQ4, mediated PHB accumulation in the mutant Ralstonia eutropha PHB(-)4 and recombinant Escherichia coli JM109(pBHR69), which produced the beta-ketothiolase and acetoacetyl-CoA reductase from R. eutropha, contributing to approximately 62% and 6% of cell dry weight, respectively. Functional expression of the coding region of phaC was confirmed by immunoblotting and in vitro PHB synthase activity.